Infection of humans and other mammals by members of the Alphavirus genus within the Togaviridae can result in fever, rash, arthritis, encephalitis and death. Several alphaviruses have been designated by the Centers for Disease Control and Prevention as Category B agents of concern for possible use in bioterrorism, and as Priority Pathogens by the NIAID. There is currently no specific treatment available for diseases caused by these pathogens. We are studying Alphavirus replication with the long-term goal of identifying potential therapeutic targets. Replication of incoming positive-sense genomic RNA, which occurs in membrane-associated complexes containing the viral nonstructural proteins (nsPs), first generates a minus strand copy, followed by new progeny plus strand RNAs. We hypothesize that in addition to known differences in the viral nsP components of the minus and plus strand replicases, that recruitment of different host factors facilitates their disparate functions. We also hypothesize that alphaviruses utilize a common strategy of host factor recruitment to replicate their respective genomes. Using mutants expressing a tagged nsP3 protein, we propose to use immunological techniques to isolate the plus and minus strand replicases from three representative alphaviruses, Sindbis virus, Ross River virus, and Venezuelan equine encephalitis virus. The host factors present in the replication complexes will be identified by proteomic techniques. Focusing on factors commonly present in the Alphavirus plus and/or minus strand replicase complexes, we will verify each factor's association with the replicase in intact cells using confocal fluorescence microscopy. Using molecular and biochemical approaches, we will assess the effects of associated host components on viral replication and map the protein-protein (or protein-RNA) interactions of the host factors. Understanding the components of and molecular interactions within the Alphavirus minus and plus strand replicases will facilitate future exploration for inhibitors of these important pathogens.